I. Field of the Invention
This invention relates to a method for stably engrafted non-bovine (xenogeneic), preferably human B- and T-cells in ungulates, and other hoofed animals such as bovines, pigs, horses, sheep, buffalo, and goats. The method of the present invention is particularly advantageous because it should result in cloned ungulates and other hoofed animals, e.g., bovines, that produce non-bovine, preferably human in lieu of endogenous antibodies. The invention more specifically relates to a method for producing IgM, Igα, E2A, EBF, BSAP, rag-1, or rag-2 knockout ungulates, that do not express endogenous immunoglobulins, which are engrafted with heterologous hematopoietic stem cells.
II. Description of the Related Art
One of the major impediments facing the development of in vivo therapeutic and diagnostic applications for antibodies in humans is the intrinsic immunogenicity of non-human immunoglobulins. For example, when immunocompetent human patients are administered therapeutic doses of rodent antibodies, the patients produce antibodies against the rodent immunoglobulin sequences; these human anti-mouse antibodies (HAMA) neutralize the therapeutic antibodies and can cause acute toxicity. Hence, it is desirable to produce human immunoglobulins that are reactive with specific antigens that are pathogenic or contribute to pathogenic conditions, or are otherwise promising therapeutic and/or diagnostic targets.
Present technology for obtaining polyclonal human antibody for use in passive immunotherapy or prophylaxis involves collection of blood from thousands of human donors, pooling it, and extracting human immunoglobulin. This technology producing human antibody or use in therapy has two major drawbacks. First, the supplies of human blood are too small to meet the demand for human immunoglobulin. Second, medical and ethical considerations preclude the deliberate immunization of human donors with a broad panel of microbes and other agents, many of which are potentially pathogenic, to assure that antibodies to these agents are present and of the highest practicable titer. There are no improvements to this current technology for obtaining polyclonal human antibody for passive immunotherapy that are likely to solve these important quantitative and qualitative problems.
Previous technology for generating monoclonal antibodies involved pre-exposing, or priming, an animal (usually a rat or mouse) with antigen, harvesting B-cells from that animal, and generating a library of hybridoma clones. By screening a hybridoma population for antigen binding specificity (idiotype) and also screening for immunoglobulin class (isotype), it is possible to select hybridoma clones that secrete the desired antibody. However, when these methods are applied for the purpose of generating human monoclonal antibodies, obtaining hybridomas that produce human antibodies of predefined specificity is a serious technological obstacle.
The construction of animals that are transgenic for various forms, rearranged and unrearranged, of human immunoglobulin genes has been used to produce human antibodies in nonhuman species.
Transgenic animals which produce foreign immunoglobulin are well known in the art. For example, Lonberg et al. (U.S. Pat. Nos. 5,814,318; 5,877,397; 5,874,299; 5,789,650; 5,770,429; 5,661,016; 5,625,126; and 5,545,806) disclose a method of producing transgenic non-human animals which produce human antibodies. The methods of Lonberg et al. involved either suppressing the endogenous immunoglobulin genes by using antisense polynucleotides and/or antiserum directed against endogenous immunoglobulins or inactivating both the endogenous light and heavy chain genes by homologous recombination. They next introduced sequences encoding the foreign immunoglobulin genes thereby producing a transgenic animal. The method of Lonberg et al. produces a variety of antibodies having various isotypes specific for a specific antigen.
Surani et al. (U.S. Pat. No. 5,545,807) also discloses a method for producing antibodies from transgenic animals. The method of Surani et al. involves using a host animal which lacks the genetic material relevant for encoding immunoglobulins. To this animal host, genetic material is added that encodes for heterologous unrearranged and rearranged immunoglobulin heavy and light chain of foreign origin capable of undergoing isotype switching in vivo. Following immunization, polyclonal antisera may be produced from such a transgenic animal. The transgenic non-human animals produced by the method of Surani et al. are able to produce, in one embodiment, IgG, IgA, and/or IgE antibodies that are encoded by human immunoglobulin genetic sequences and which also bind specific human antigens with high affinity.
DeBoer et al. (U.S. Pat. No. 5,633,076) and Meade et al. (U.S. Pat. No. 5,849,992) both disclose the production of transgenic cows which produce antibodies in their milk. DeBoer et al. produce transgenic cows by introducing a transgene, encoding an antibody gene operably linked to a mammary specific promoter, into a cow zygote. Meade et al. produce transgenic mammals which express antibodies in their milk by introducing downstream of a mammary specific promoter foreign DNA segments encoding specific paired immunoglobulin heavy and light chains.
However, the use of transgenics to produce domestic animals that express human antibodies for passive immunotherapy requires the solution of a number of problems. These include the levels at which human antibody transgenes might be expressed in non-human hosts, their ability to undergo class switching, affinity maturation and the immunogenicity in humans of inappropriately glycosylated human antibody. These problems stem from the introduction and expression of human antibody genes in non-human cells. A system that would allow for the introduction of human hematopoietic stem cells into non-humans, especially large animals of agricultural interest such as bovines and other ungulates (e.g., cattle, sheep, or goats), and their development into immunocompetent human B-cells would provide a comprehensive solution of these problems.
However, the immune system poses a major barrier to the introduction of foreign hematopoietic stem cells into an animal of another species. With respect to this barrier, it has been reported that the immune system can potentially be disabled by targeted disruption of rag-1 or rag-2 (recombinase activating gene) (hereinafter rag-1 knockout or rag-2 knockout). (See, e.g., Martin et al., J. Clin. Endocrinol. 79(3):716-723 (1994); Mazurier et al., J Interferon Cytokine Res. 19(5):533-541 (1999); and Goldman et al., Br. J. Haematol. 103(2):335-342 (1998)). Also, the production of IgM knockout mice that do not express functional endogenous B-cells have been reported. (See, Ehrenstein et al., Proc. Natl. Acad. Sci., USA 95(17):10089-10093 (1998); and Erlandsson et al., Eur. J. Immunol. 28(8):2355-2365 (1998)). Rag-1 or rag-2 knockout animals potentially are unable to conduct the gene rearrangements that are necessary to generate the antigen receptors of B or T lymphocytes. Consequently, they do not develop native B- or T-cells. Moreover, because these animals do not produce B and T lymphocytes, the use of rag-1 or rag-2 knockout mice for engraftment of human hematopoietic stem cells has been reported.
Particularly, such a system has been developed in mice, wherein human hematopoietic progenitor cells have been added to rag-2 knockout mice. Yahata et al., Immunol. Lett. 62(3):165-170 (1998) discloses transferring IL-12-induced splenic hematopoietic progenitor cells into rag-2 knockout mice to reconstitute their immune system. This resulted in the production of mice having stably engrafted therein both human B- and T-lymphocytes. However, while the development of human B- and T-lymphocytes in mice has been reported, there has been no report of human or other heterologous species hematopoietic stem cells stably engrafted into an ungulate or any indication that such cells, if stably engrafted will begin to develop into fully immunocompetent B- and T-cells when implanted into ungulates that do not produce B-cells because of a genetic modification, e.g., IgM, Igα, EIA, BSAP, EBF, rag-1, or rag-2 knockout animals other than mice, and more specifically large agricultural animals such as cattle and other ungulates.
While it is anticipated that ungulates will be able to become stably engrafted with human stem cells and provide for the development of xenogeneic immunocompetent B- and T-cells in ungulates and other hoofed animals for which endogenous antibody production has been knocked out, e.g., by knockout of IgM, rag-1, or rag-2 gene, this outcome may not be feasible for various reasons. For example, natural killer cells do not depend on the rearrangement of antigen receptor genes for their cell killing activities. Consequently foreign lymphocytes, e.g., human lymphocytes potentially may be attacked by endogenous natural killer cells and thereby prevent the establishment of human B- and T-cells populations in B-cell deficient ungulates, e.g., IgM, rag-1, or rag-2 deficient animals (provide for stable engraftment). Furthermore, the manner by which B-cells and antibodies develop in humans is quite different from, for example, cattle or other ungulates. In humans, B-cells arise in bone marrow and the primary repertoire is diversified by extensive rearrangement and junctional diversity. By contrast, in cattle, bone marrow is not the site of B-cell origin. Primary repertoire diversification takes place in the spleen and gut associated lymphoid tissue rather than in bone marrow. Also, repertoire diversification in cattle uses relatively few rearrangements and little junctional diversity. Most of the diversity seen in the primary repertoire is the result of massive, variable region focused somatic mutation of rearranged genes. The sharp differences in B-cell development and primary repertoire development between humans and cattle makes it unpredictable whether a functional and diverse repertoire of human B-cells will develop from human hematopoietic stem cells transplanted into cattle and other ungulates and hoofed animals.
Furthermore, until now, various technical barriers have prevented the creation of ungulates, and other large agricultural animals, e.g., cattle, sheep, horses, goats, and buffalo, that have been genetically manipulated in order to knockout antibody production, e.g., by genetically knocking out B-cell production and optionally T-cell production. Particularly, the use of conventional protocols for obtaining double knockouts in primary cell lines with limited life spans in culture is uncertain and difficult. The present inventors propose a method that should overcome these barriers and provides a protocol for producing ungulates having a double knockout that prevents B-cell formation, e.g., E2A, EBF, BSAP, IgM, rag-1, and rag-2 knockout ungulates, especially cattle which have stably engrafted foreign B- and T-lymphocytes, preferably human, canine, feline, rat, or murine, and which produce foreign immunoglobulins in their serum of the species of origin of the particular engrafted hematopoietic stem cells.